Targeted delivery of bioaffecting compounds for the treatment of cancer

ABSTRACT

A homogeneous conjugate for targeting and treating diseased cells wherein the conjugate comprises an anticancer drug and a targeting protein, wherein said anti-cancer drug is selected from the group consisting of heat sensitizers, photosensitizers and apoptosis inducing compounds, a method for making such a conjugate, and methods for using the conjugate. The targeting protein is preferably transferrin.

FIELD OF THE INVENTION

[0001] This invention relates generally to the field of bio-affectingmaterials useful in the treatment of diseased cells, such as cancercells and, more specifically to a conjugate of a targeting agent and abio-affecting material, where the bio-affecting material is a heatsensitizer, a photosensitizer or an apoptosis inducing compound.

BACKGROUND OF THE INVENTION

[0002] Two of the most devastating problems in cancer treatment aredrug-toxicity, which debilitates patients, and drug-resistance, which isnormally countered with even higher drug dosages and thus amplifies theproblem of drug-toxicity, often resulting in death. One way to solve theproblem of drug-toxicity is to target drugs for delivery only to cancercells. Many researchers are working to develop antibodies to deliverdrugs to targeted cells, and this approach holds promise, but antibodiesare not without problems. For example, antibodies often bind to normaltissues, and they also can damage blood vessels (e.g., vascular leaksyndrome) and cause dangerous allergic reactions (e.g. anaphylaxis).

[0003] Research is also progressing in connection with the use ofconjugates of transferrin and doxorubicin, daunomycin, methotrexate,vincristin, 6-mercaptopurine, cytosine arabinoside, cyclophosphamide,and radioiodine as described in U.S. Pat. Nos. 5,108,987; 5,000,935;4,895,714; and 4,886,780. The inventions described in these patents doesnot use antibodies. Instead, it uses a protein found in normal humanblood. This protein is transferrin, which delivers iron. Normal cellsrarely require iron, but cancer cells require large amounts of iron tomaintain their pathologically increased rates of metabolism. Becausecancer cells require more iron, they have transferrin receptorssubstantially permanently on their surfaces, whereas normal cells donot. These inventions exploit these receptors by administeringanticancer drugs conjugated with transferrin, which delivers the drugssubstantially only to the surface of cancer cells.

[0004] Drug targeting spares normal cells, requires less drug, andsignificantly diminishes drug-toxicity. In contrast, when anticancerdrugs are administered without being targeted, they kill normal cells aswell as cancer cells. They are particularly toxic to cells of the immunesystem and to the system responsible for blood clotting. Thus,infections and bleeding are principal complications of chemotherapy incancer patients. These complications require expensive services,hospitalizations, intensive care, and life-support systems, which areuncomfortable and expensive for the patient. These problems are largelypreventable by using targeted delivery systems.

[0005] The problem of drug-toxicity consumes huge blocks of doctors' andnurses' time, and is responsible for much of the cost of cancer care.For example, it is commonly understood that about 70% of calls tooncologists relate to a problem of drug-toxicity. Today there is nosatisfactory way to treat drug-toxicity, except to use less drug. In theabsence of targeted delivery the use of less drug is counterintuitive inthe case of drug resistant cancers. Targeted delivery allows the use ofless drug, because more of the administered drug is deliveredspecifically to cancer cells rather than being nonspecificallydistributed around the body. In this sense, targeted delivery is likeshooting with a rifle, while conventional delivery is like shooting witha shotgun. A solution to the problem of drug-toxicity will dramaticallytransform chemotherapy in cancer patients. It is a purpose of thisinvention to reduce such adverse effects of chemotherapy.

[0006] The problem of drug-resistance is equally as serious as theproblem of drug-toxicity. This problem is typified by a patientdiagnosed with cancer who is treated and responds with a symptomlessremission that lasts many months, and who later sees the cancer returnsin a form that no longer responds to any known drug. This scenario ofdrug-resistance is all too common, Yet today there is no satisfactorysolution, except the use of larger amounts of more powerful drugs, thatin turn can cause serious drug-toxicity problems, often resulting indeath. Thus, a solution to the problem of drug-resistance wouldsignificantly diminish the problem of drug-toxicity. A major effort wasdevoted to the use of P-glycoprotein inhibitors such as verapamil (Ford,Hematol/OncolClin N Am 1995; 9:337), cyclosporine (Bartlett et al, JClin Oncol 1994; 12:835) and cyclosporine derivatives such as SDZ PSC833 (Kusunoki et al., Jpn J Cancer Res 1999; 89:1220), and tamoxifen(Pommerenke et al., J Cancer Res Clin Oncol 1994; 120:422), but theseapproaches have not been clinically satisfactory because they introducednew problems in the pharmacokinetics of chemotherapy (Sikic et al.,Cancer Chemother Pharmacol 1997; 40:S13). Other approaches are thedesign of MDR-reversing drugs (Naito & Tsuruo, Cancer ChemotherPharmacol 1997; 40:S20); the use of P-glycoprotein antisenseoligonucleotides (Bertram et al, Anti-Cancer Drugs 1995; 6:124); the useof retrovirus-mediated transfer of anti-MDR ribozymes (Wang et al.,Human Gene Therapy, 1999; 10:1185), and the design of chemotherapy drugsthat are not removed from cancer cells by MDR or MRP pumps (Mankhetkomet al., Mol Pharmacol 1996; 49:532), but none of this research hasprovided a solution to the clinical problem of drug resistance (Arceci,Br J Haematol 2000; 110:285). Another approach is to circumvent theMDR/MRP pumps by delivering anti-cancer drugs as conjugates of largermolecules such as albumin (Ohkawa et al., Cancer Res 1993; 53:4238),alpha-fetoprotein (Moskaleva et al., Cell Biol Int 1997; 21:793) anddextran (Fong et al., Anticancer Res 1996; 16:3773). Although these arevariously effective at evading experimental drug resistance, they areunproven in patients. Protein-targeted drug delivery can overcome theproblem of drug-resistance. Thus, another purpose of the presentinvention is to resolve the issue of painful and expensive deaths fromdrug-resistant cancers.

[0007] The effectiveness of proteins conjugated with bio-affectingmolecules has been demonstrated and is described in the US patentsmentioned above. It has been determined; however, that the efficiency ofsuch conjugates ill treating stressed cells, such as cancer cells, isreduced by the presence of agglutinated conjugates or by the presence ofconjugates of a bio-affecting molecule with protein fragments or withtwo or three protein molecules and is greatly enhanced when the proteinto bio-affecting molecule ratio is closer to 1:1 or 1:2, depending onthe bio-affecting molecule. Obtaining conjugates of higher efficiencyhas, in the past, been a slow, tedious and expensive process thatrequires separating a fraction of conjugate having the desired averageratio of bio-affecting molecule to protein from a larger samplecomprising such molecules conjugated with protein fragments, with aplurality of proteins and proteins conjugated with a plurality ofbio-affecting molecules. Using homogeneous protein-drug conjugates inwhich the protein component carries a predetermined number ofbio-affecting molecules can more effectively kill both drug-resistantand drug-sensitive cancer cells. In the past the expense andinefficiency inherent in producing useful conjugates in a useful volumehas been a problem for the commercialization of such conjugates and fortheir widespread use in medicine. There is a need for a substantiallyhomogeneous drug-protein conjugate and for a method of making such aconjugate that is more efficient, more precise and less costly. It isone purpose of this invention to provide such a homogeneous conjugatemade by a more efficient method.

DESCRIPTION OF THE RELATED ART

[0008] The first report of transferrin receptors on human cancer cellswas by Faulk and colleagues in 1980 (1). This was followed by manyreports of transferrin receptors in different types of human cancers(2), as seen in the following Table. Tumor Studied References Breast  1,3 Leukemia  4, 5 Lung  6 Brain  7 Liver  8 Bladder  9 Gastrointestinal10 Ovary 11 Non-Hodgkin's lymphoma 12 Lymphoma/melanoma 13, 14Nasopharyngeal 15 Cervix 16

[0009] Transferrin Receptors on Normal and on Cancer Cells.

[0010] No single study has asked if all human cancers have up-regulatedtransferrin receptors, or if all normal cells have down-regulatedtransferrin receptors, but data from many quarters suggest that theanswer to both questions is yes. For example, immature erythrocytes(i.e., normoblasts and reticulocytes) have transferrin receptors ontheir surfaces, but mature erythrocytes do not (17). Circulatingmonocytes also do not have up-regulated transferrin receptors (8), andmacrophages, including Kupffer cells, acquire most of their iron by atransfenrin-independent method of erythrophagocytosis (19). In fact, invivo studies indicate that virtually no iron enters thereticuloendothelial system from plasma transferrin (for review, seereference 20). Macrophage transferrin receptors are down regulated bycytokines such as gamma interferon (21), presumably as a mechanism ofiron-restriction to kill intracellular parasites (22).

[0011] In resting lymphocytes, not only are transferrin receptors downregulated, but the gene for transferrin receptor is not measurable (23).In contrast stiulated lymphocytes up regulate transferrin receptors inlate GI (24). Receptor expression occurs subsequent to expression of thec-myc proto-oncogene and following up-regulation of IL-2 receptor (25),and is accompanied by a measurable increase in iron-regulatory proteinbinding activity (26), which stabilizes transferrin receptor mRNA (27).This is true for both T and B lymphocytes (28), and is an IL-2-dependentresponse (29).

[0012] Cell stimulation resulting in the up regulation of receptors fortransferrin is known to result from stress experienced, for example, bycells invaded by a viral or protozoan factor and by cancer cells.

[0013] Up-and-down regulation of transferrin receptors for normal andtumor cells has been shown by studies of antigen or lectin stimulation(i.e., receptor up-regulation), and by studies of differentiation models(30-33) using retinoic acid (i.e., receptor down-regulation). Base-linedata from these experimental models suggest that these receptors aredown-regulated from the plasma membranes of most normal, adult, restinghuman cells (34). Exceptions are the circulatory barrier systems, whichinclude the materno-fetal barrier with its transferrin receptor-richsyncytiotrophoblast (35); the blood-brain barrier with its transferrinreceptor-rich capillary endothelial cells (36); and, the blood-testisbarrier with its transferrin receptor-rich Sertoli cells (37).

[0014] Transferrin-Drug Conjugates in Laboratory Animals

[0015] The efficacy of transferrin-drug conjugates has been investigatedin several animal models. For example, conjugates of transferrin withdiphtheria toxin decreased xenografted gliomaas in nude mice by 95% onday 14, and the gliomas did not recur by day 30 (74). Also,glutaraldehyde-prepared transferrin-doxorubicin conjugates have beenfound to rescue nude mice from death by human mesothelioma cells,significantly prolonging life compared to animals treated only withdoxorubicin (75). In addition, transferrin has been coupled to herpessimplex thymidine kinase by using biotin-streptavidin technology, andthese conjugates significantly prolonged life in nude mice inoculatedwith metastasizing K562 tumor cells (76). Finally, the maximum tolerateddose of human transferrin-doxorubicin conjugates in nude mice has beenfound to be 20 mg/kg (iv) for conjugates and only 8 mg/kg (iv) for freedrug (41).

[0016] Transferrin-Drug Conjugates in Human Patients

[0017] There are two clinical reports of transferrin-drug conjugates.The first, published in 1990, was a preliminary study of seven acuteleukemia patients treated intravenously with 1 mg/day ofglutaraldehyde-prepared transferrin-doxorubicin conjugates for 5 days.With these low doses, there were no toxic effects and the number ofleukemic cells in peripheral blood of the 7 patients decreased by 86%within 10-days following therapy (77). In addition, there was noextension of disease as assessed by examination of bone marrow biopsiesbefore and after treatment.

[0018] The second, published from the NIH in 1997, involved 15 patientswith recurrent brain cancers treated with thioether-bonded transferrinconjugates of a genetic mutant of diphtheria toxin (44). The conjugateswere delivered by high-flow interstitial microinfusion, which has beenshown to produce effective pefusion of radiolabeled transferrin inprimate brains with minimal inflammatory responses (78). Magneticresonance imaging revealed at least a 50% reduction in tumor volume in 9of the 15 patients, including 2 cases of complete remission (44).

[0019] There is an unpublished clinical study of 23 patients withadvanced ovarian cancer who were randomized into test (12 patients) andplacebo (11 patients) groups. The test group receivedtransferrin-doxorubicin conjugates equivalent to 1 mg doxorubicin perday on days 15 through 19 of monthly treatment cycles. A significantdifference was revealed by Cox regression estimates of survival ratesfor patients treated with transferrin-doxorubicin conjugates when thetime between diagnosis and randomization was 18 months.

[0020] Another unpublished study is a 22-year old male with metastaticdisease from a sarcoma of his right atriunm who was treated byconventional protocols without response.

[0021] His lungs were filled with metastatic lesions when his physicianfather obtained an IND from the FDA for the use oftransferrin-doxorubicin conjugates, and treatment was begun in August,2000. By November, the lungs were substantially cleared of metastaticlesions, and by January there was no radiological evidence of tumor. Hepresently (May 2002) is active, receiving only transferrin-doxorubicin.

[0022] The targeted delivery of drugs has the advantage of increasingefficacy while using less drug, thereby decreasing toxicity and causingless damage to normal cells, all of which effectively decrease costs andincrease the quality of patient care. Targeted delivery also avoidsdrug-resistance, which is activated by the non-specific entrance ofdrugs into cells (79). Because transferrin-drug conjugates can entercells specifically by employing a receptor-specific pathway (80,81),they are trafficked around drug-resistance mechanisms, such as effluxpumps in resistant cells.

[0023] It was reported in 1992 that transferrin-doxorubicin conjugateseffectively kill multi-drug resistant cells (82). This finding wasconfirmed in 1993 (83), and was extended to several types ofdrug-resistant cells in 1994 (84), 1996 (85) and 2000 (86).Interestingly, doxorubicin immobilized on solid carriers also has beenshown to be effective against doxorubicin-resistant cells (60,87). Infact, a concept is emerging that vectorization of doxorubicin with oneof several peptide vectors is effective in overcoming multi-drugresistance (88).

[0024] Preparation of Transferrin-Drug Conjugates.

[0025] A method for the preparation of transferrin-doxorubicinconjugates was published first m 1984 (38), following which there havebeen many reports of methods for the preparation of transferrin-drugconjugates, some of which are listed in the following Table. TransferrinLabel Method Used References Doxorubicin Glutaraldehyde 38, 39, 40Doxorubicin Maleimide 41 Mitomycin C Glutaryl Spacer 42 NeocarzinostatinSuccinimide 43 Diphtheria Toxin Thioester 44 Chlorambucil Maleimide 45Paclitaxol Glutaraldehyde 46 Daunorubicin Glutaraldehyde 47 TitaniumCarbonate 48 Insulin Disulfide 49 Gallium Carbonate 50 PlatinumMethionine 51 Saporin/ricin Succinimide 52 Ruthenium Bicarbonate 53Growth Factor Fusion Protein 54 HIV Protease Recombinant 55

[0026] Transferrin conjugates of doxorubicin havebeen prepared by usingglutaraldehyde-mediated Schiff base formation (56,57), which forms anacid-resistant bond between epsilon-amino lysine groups of transferrinand the 3′amino position of doxorubicin Such conjugates of doxorubicincan kill cancer cells through a plasma membrane-mediated mechanisms (forreview, see reference 58). Although DNA intercalation is an establishedmechanism of cell death by doxorubicin, immobilized doxorubicincorrugated with proteins capable of binding with receptors on the cellsurface activate plasma membrane-mediated mechanisms to kill cells(59,60). It thus appears that conjugates of doxorubicin with transferrinkill cells by activating plasma membrane-mediated mechanisms thatinvolve both doxorubicin and transferrin receptors.

SUMMARY OF THE INVENTION

[0027] It is apparent that targeting agent-drug conjugates have ageneric possibility of changing how drugs are delivered, as well as aspecific possibility of changing how drugs are delivered to cancerpatients. In the present invention, non-antibody proteins such astransferrin, ceruloplasmin, vitamin binding proteins such astranscobalamin, hormones, somatostatin, cytokines, low densitylipoproteins, growth factors, growth factor-like molecules, are used astargeting agents in combination with anti-cancer compounds. Becausetransferrin and transcobalamin are excellent carriers for deliveringcompounds into drug-sensitive and drug resistant cancer cells, thetargeted delivery system in the present invention preferably consists oftransferrin or transcobalamin coupled to anticancer compounds selectedfrom the group consisting of heat sensitizers; photosensitizers andapoptosis inducing confounds.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to conjugates with a targetingagent and an antitumor agent. The targeting agents according to thepresent invention include butare not limited to transferrin,ceruloplasmin, somatostatin, vitamins, vitamin binding proteins such astranscobalamin, hormones, cytokines, low density lipoproteins, growthfactor-like molecules, folic acid-like molecules and growth factors. Theanti-tumor agents include but are not limited to heat sensitizers suchas hematophorphyrine and low-dose verapamil, apoptosis inducingcompounds such as deferoxamine, and photosensiers such as porfimersodium, metatetrahydroxyphenylchlorin, and hematophorphyrin derivatives.Preferably the targeting agent is transferrin or transcobalamin.Preferably the anti-tumor agent and the targeting agent are conjugatedby means of a linker. Suitable linkers include but are not limited toglutaraldehyde (Bérczi et al., Arch Biochem Biophys 1993; 300: 356),disulfide coupling (Sash et al., Jap J Cancer Res 1993; 84: 191) andbenzoyl hydrazone (Kratz et al., J Pharm Sci 1998; 87: 338).

[0029] Transferrin-Heat Sensitizer Conjugates

[0030] Hyperthermia is widely used in cancer treatment either alone orin combination with chemotherapy and radiotherapy (Kong et al., Int JHyperthermia 1999; 15: 345; Raaphorst et al., Oncology Reports 1998; 5:971; Mager et al., Anticancer Res 1999; 19: 3403). In combinationtreatments, hyperthermia (41-45° C.) has been shown to be an effectiveradiation sensitizer (Sukurai et al., Int J Rad Biol 1999; 75: 739;Harima et al., Cancer 2000; 88: 132). Heat enhances the cytotoxic effectof certain anticancer agents such as bleomycin (Khadir et al., ArchBiochem Biophys 1999; 370: 163), carboplatin (Steller et al., CancerChemother Pharmacol 1999; 43: 106), doxorubicin (Kawai et al., Cancer1997; 79: 214) and cisplatin(Raaphoist etal., Oncol Reports 1998; 5:971; Benoit et al., Chirurgie 1999; 124:375). Hyperthermia is used insome therapy-resistant malignancies (Wessalowski et al., Int JHyperthermia 1999; 15: 455) and high temperature hyperthermia (greaterthan 50° C.) has been used for selective tissue destruction as analternative to conventional invasive surgery (Diederich et al.,Ultrasound In Med Biol 1999; 25: 871).

[0031] The effectiveness of hyperthermia is strongly dependent on theability to localize and maintain therapeutic temperature elevations(Diederich et al., Ultrasound In Med Biol. 1999; 25: 871). To achievebetter results, several techniques have been used to produce controlledtemperature increases in tissue. Some of these tecbniques involve theuse of YAG Lasers (Graeber et al., Laryngoscopy 1999;109: 447),radiofrequences (Wust et al., Int J Hyperthermia 1998;14: 459),ultrasound (Diederich et al., Ultrasound In Med Biol 1999; 25: 87 I),and microwaves (Finger, Ophthalmol 1997;104:1794). Hyperthermia is moreeffective at high temperatures, but its use is limited by the need toprotect healthy tissues from heat damage.

[0032] Heat induces toxic characteristics in several compounds that arenot toxic at normal body temperature. An example of this ishematophorphyrine. Ai 41.5° C. this molecule is a potent heat (orhyperthermia) sensizer, and its ability to sensitize increases in adose-dependent mnniler (Saito et al., Int J Hyperthermia 1998;14: 503).Another example of a heat (or hyperthermia) sensitizers is the use oflow-dose verapamil at 42° C. (Shchepotin et al., Int J Hyperthermia1997;13: 547). Heat sensitizers do not elevate local temperatures. Theyfunction by producing increased local toxicity in the presence of heat.If non-toxic compounds that produce toxicity at higher than bodytemperature, or toxic compounds that increase their toxicities atelevated temperature, could be delivered only to cancer cells, thenhyperthermia could be used more efficiently to treat cancer. Such atargeted delivery system would not damage healthy tissues, which wouldreceive only the effects of elevated temperatures.

[0033] This invention introduces a way of selectively deliveringcompounds that are highly toxic for cancer cells at increasedtemperatures. This targeted delivery system involves the use oftransferrin as the carrier of the heat (or hyperthermia) sensitizer.

[0034] The use of targeted delivery of heat sensitizers would make theoutcome of hyperthermia more effective by itself or in combination withother treatments, since heat sensitive toxicity can be concentrated incancer cells. In this way, damage of healthy tissues is minimal, as mostsensitizers can work at relatively low temperatures (e.g., 41-42° C.).

[0035] Since transferrin is an excellent carrier for deliveringcompounds to drug-sensitive and drug resistant cancer cells, thetargeted delivery system in this invention consists of transferrincoupled to heat sensitizers. Transferrin for use in human patients iscommercially available (for example, from the Finish Red Cross). Severalcoupling processes such as glutaraldehyde coupling (Berczi et al., ArchBiochem Biophys 1993; 300: 356), disulfide coupling (Sash et al, Jap JCancer Res 1993; 84: 191) and benzoyl hydrazone coupling (Kratz et al.,J Pharm Sci 1998; 87: 338) have been used to couple transferrin withother compounds. The broad variety of coupling procedures allow theconjugation of a wide range of sensitizers to transferrin, resulting ineither permanent or dissociable bonding of sensitizers with thetransferrin molecule. Following the conjugation of transferrin to theselected heat sensitizer, the conjugates should be separated from theuncoupled components, for example by using chromatographic and/ordialysis techniques. Examples of suitable heat sensitizers include butare not limited to doxorubicin, cisplatin and hematophosphrine.

[0036] Transferrin-Photosensitizer Conjugates

[0037] Photochemotherapy of cancer often is called photodynamic therapy(PDT). This form of therapy arose in the late 1970s and appeared as analternative form of treatment for localized inoperable lesions(Dougherty et al., Cancer Res 1978; 38:2628). It involves theadministration a tumor-localizing agent (Hayata et al., Chest 1982;81:269), which may require metabolic changes (e.g., a prodrug), followedby activation of the agent by light of a specific wavelength (Sharman etal., Meth in Enzymol 2000; 319:376). This results in production of asequence of photochemical and photobiological reactions, such as theproduction of singlet oxygen (Yuan et al., Radiation Res 1997; 148:386)that initiate tumor necrosis (Ochsner, J Photochem Photobiol 1997,39: 1) via thrombosis (Henderson & Dougherty, J Photochem Photobiol1992; 55:145) and apoptosis (Luo & Kessel, Biochem Biophys Res Corn1996; 221:72).

[0038] Progress in PDT during the 1990s has included development of moreefficient photosensitizers that absorb at longer wavelengths and requireless energy for activation, thus allowing deeper penetration. New fiberoptics also have been developed that allow illumination of difficultgeometric configurations, and smaller, less expensive and more mobilelasers have become more readily available. This research and developmenthas allowed PDT to evolve into a more widely used therapeutic modality,both for the primary treatment of cancer and for an adjunct to surgicaldebulking of larger tumors Treatment with PDT has been used in a widevariety of cancers, including basal cell carcinomas (Stefanidon et al.,Euro J Dermatol 2000; 10:351), lymphomas (Orenstein et al., DermatolSurg 2000; 26:765), stomach cancer (Gassner et al., GastrointestEndoscop Clin N Am 2000; 10:461) and leukemia (Danilatou et al.,Leukemia Res 2000; 24:427). Indeed, a recent review lists more than 60types of cancer that have been treated with the FDA-approvedphotosensitizer Photofrin (porfimer sodium). In addition to its use incancer treatment, PDT also has been used in Dermatology (Hongcharu etal., J Invest Dermatol 2000; 115:183), Ophthalmology Tannous et al,Invest Ophthal Visual Sci 2000; 41:749), and recently PDT has been usedin angioplasty procedures (Rockson et al, Circulation 2000; 102:591).

[0039] The first photosensitizer to receive FDA approval was Photofrin,produced by QLT Photo Therapeutics in Canada Another widely studiedphotosensititer is aminolevulnic acid. Other molecules in clinicaltrials are SnET2, which is a chlorin photosensitizer produced asPurlytin by Pharmacia/Upjohn. Another molecule is mTHPC produced byScotia Pharmaceuticals as Foscan, which is ametatetrahydroxyphenylchlorin. Macular degeneration has been inhibitedby using the photosensitizer Verteporfin, which is a haematophorphyrinderivative. These and several other molecules used as photosensitizershave been reviewed by Stewart and colleagues (Radiother Oncol 1998;48:233) and by Dougherty and colleagues (J Natl Cancer Inst 1998;90:889).

[0040] Since the presence of transferrin receptors is restrictedprincipally to the surface of tumor cells, as originally described byFaulk and colleagues in the Lancet (1980; 2:390), the use of transferrinas the carrier of photosensitizers virtually assures that the drug willbe targeted only to cancer cells, thus allowing the effects of PDT to belocalized to the tumor and only to the tumor. In addition to minimizingcollateral damage of normal cells, targeting of the photosensitizerdrugs as conjugates of transferrin allows the use of lower doses ofdrugs, which will diminish the side effects of some drugs. For example,solar photosensitivity is a dose-related problem encountered in somepatients treated with Photoprin.

[0041] The present invention of targeting the delivery ofphotosensitizer drugs as conjugates of transferrin to transferrinreceptors on the surface of cancer cells has several advantages thatderive from the biology of the carrier. The foremost of these is theproperty of transferrin drug conjugates to cross the blood-brain-barrier(Broadwell et al., Exp Neurol 1996; 142:47). This property is endowed inthe molecule by the normal presence of transferrin receptors on theluminal surface of endothelial cells that provide the microcirculationof the blood-brain-barrier (Moos & Morgan, Cell Mol Neurobiol 2000;20:77). This has been utilized to deliver molecules from the bloodcirculation into the brain as conjugates of transferrin (Park et al., JDrug Target 1998; 6:53), which allows for the targeted use of PDT inpatients with brain cancer. Another biological property of transferrinthat positions it as an ideal carrier for photosensitizer drugs is thatit is endocytosed into cells after being bound by transferrin receptors(Berczi et al, Eur J Biochem 1993; 213:427); This property can localizephotosensitizers to the inside of cancer cells, which concentrates thedrug and augments killing. In this regard, it is relevant thatdoxorubicin conjugates of transferrin have been used to killdoxorubicin-resistant cancer cells that were not killed by 10-foldhigher concentrations of free doxombicin (Fritzer et al., Int J Cancer1992; 52:619).

[0042] In the following discussion transferrin is illustrative of thetargeting protein Transferrin can be isolated from normal blood plasma,purchased from any of several companies, including both the American andFinnish Red Cross, or be prepared from recombinant technology (Ali etal., J Biol Chem 1999; 274:24066). To form a drug:transferrin conjugate,the transferrin molecule must be modified in such a way as to prepare itto be coupled with a photosensitizer drug. For example, transferrin mustbe thiolated in order to form maleimide derivatives of drugs (Kratz etal., J Pharm Sci 1998; 87:338). The exact method of conjugation willdepend upon the chemistry of the photosensitizer to be coupled withtransferrin. For example, Protofrin is a polyporphrin containing esterand ether linkages, suggesting one type of carrier (Dubowchik & Walker,Pharmacol & Therap 1999; 83:67), other drugs contain NH2-groups that canbe coupled to transferrin by using bifunctional reagents such asglutaraldehyde, and others require disulfide coupling (Sasaki et al.,Jap J Cancer Res 1993; 84:191). The wide variety of coupling proceduresallows the conjugation of a broad range of photosensitizer drugs withtransferrin, resuting in either permanent or dissociable bonding ofdrugs with the transferrin molecule (Barabas et al., J Biol Chem 1992;267:9437). Whatever procedure is used, after the coupling reaction thedrug-protein conjugates must be separated from uncoupled drug and/orfree protein, preferably by using chromatography or dialysis techniques.

[0043] Transferrin-Apoptosis Inducing Compound Conjugates

[0044] A mechanism for iron-restricted cytotoxicity in cancer is thepotentiation of programmed cell death. Deferoxamine has been found toincrease arabinoside-rnediated apoptosis of human myeloid leukemia cells(Leardi et al., Br J Haematol 1998; 102:746). There is considerableevidence that a major pathway for apoptosis involves calcium-mediateddown-stream signaling subsequent to ligand-binding of programmed celldeath surface receptor CD95 (Kass & Orrenius, Environ Health Perspec1999; 107(Suppl 1):25). Similarly, the transferrin receptor functions asa signal-transduction molecule for its own recycling by increasingintracellular free calcium concentrations (Sainte-Marie et al., Europ JBiochem 1997; 250:689). Taken together, these apparently unrelatedobservations indicate a hitherto undescribed relationship betweencalcium, iron and transferrin receptors in drug resistant cancer cells.

[0045] In order to test the relationship between calcium and transferrinreceptors, the effect of calcium channel inhibitors on the regulation oftransfernin receptors was studied in drug-sensitive and drug-resistantcancer cells. The results of these experiments show that both organic(e.g. verapamil) and inorganic (CdCl₂) calcium channel blockers causedifferential effects on the regulation of transfernin receptors. Forexample, both types of channel blockers initiated down-regulation ofreceptors, but more down-regulation was observed on drug-resistant cellsthan on drug-sensitive cells. Since the half-life of down-regulation wasabout eight hours, the decrease of surface receptors did not result frominhibition of exocytosis. The addition of free iron or transferrin-boundiron also had a differential effect on the down-regulation oftransferrin receptors. The effect was similar but even more pronounced,namely iron down-regulated receptors much more on drug-resistant cellsthan on drug-sensitive cells, and produced about the same half-life ascalcium channel blockers. The addition of both iron and calcium channelblockers had no effect on cell viability.

[0046] In light of the above findings, another series of experimentswere done to determine whether the restriction of iron by deferoxaminehad differential effects on drug-sensitive and drug-resistant cancercells. The results of these experiments revealed that low concentrationsof deferoxamine up-regulated transferrin receptors on bothdrug-sensitive and drug-resistant cells, but increasing concentrationsof deferoxamine decreased transferrin receptor expression bydrug-resistant cells, and viability experiments showed that the higherconcentrations killed drug-resistant but not drug-sensitive cells; Forexample, after 48 hours in a given concentration of deferoxamine,drug-sensitive cells showed 93% viability, but only 16% ofdrug-resistant cells were alive. In summary, these three different typesof experiments revealed a marked instability of transferrin receptorregulation in drug-resistant cells, and showed that iron depletionrapidly caused death of the drug-resistant cells.

[0047] The present invention is a way to target and kill drug-resistantcells in cancer patients. In a preferred embodiment, a conjugate of adrug carrier that targets an iron chelator to drug-resistant cancercells is prepared. Transferrin is a normal blood protein that is wellsuited to deliver an iron chelator, because tumor cells have transferrinreceptors on their surface and normal, adult, resting cells do not(Berczi et al., Arch Biochem Biophys 1993; 380:356). There are many ironchelators that can be conjugated with transferrin and delivered todrug-resistant cancer cells (Tsafack et al, Mol Pharmacol 1995; 47:403),but the chemistry of deferoxamine renders it well suited for conjugationto transferrin (Tsafack et al, J Lab Clin Med 1976; 127:574). Severalconjugation or coupling procedures are possible to couple the targetingagent with other compounds, including but not limited to glutaraldehyde(Yeh & Faulk, Clin Immunol Immunopathol 1984; 32:1), disulfide coupling(Sasaki et al., Jap J Can Res 1993; 84:191) or benzoyl hydrazonecoupling (Kratz et al., J Pharm Sci 1998; 87:338). The wide variety ofcoupling procedures allows the conjugation of a broad range ofiron-chelating drugs to targeting proteins, resulting in eitherpermanent or dissociable bonding of cytotoxic drugs with the proteinmolecule (Barabas et al., J Biol Chem 1992; 267:9437); Following thecoupling reaction, drug-protein conjugates can be separated fromuncoupled drug and free protein, preferably by using chromatographic ordialysis procedures.

[0048] Technical details of the conjugation procedure can vary, but theconjugates must be active in binding and killing experiments with cancercells, and should not bind or kill significant numbers of normal cells.In light of these requirements, the preferred method for preparing theconjugates according to the present invention is the following process:

[0049] The synthesis of large amounts of homogeneoustransferrin-doxorubicin conjugates with predetermined molecular ratioswas done stoichiometrically by employing the only amino group ofdoxorubicin (DOX), which is at the 3′ amino position, to react with oneof the two reactive groups on glutaraldehyde (GLU) by drop-wise additionof a saline solution of DOX into a saline solution of GLU containing asolvent such as DMSO or another suitable cryopreservative, to a finalconcentration of a 1:1 molar ratio of DOX-to-GLU. The resulting solutionof DOX-GLU was stirred three hours at room temperature in the dark.

[0050] The molarities of DOX and GLU were the same in the above reactionin order to produce a final solution of DOX-GLU that contains neitherfree DOX nor free GLU. However, there is the possibility of free GLU insolution if one GLU reacts with two DOX to produce DOX-GLU-DOX, but thispossibility is minimized by the mass action kinetics generated bydrop-wise addition of monovalent DOX into the solution of bivalent GLU.The volumes of these reactants are not restricted, so large amounts ofhomogeneous DOX-GLU can be prepared.

[0051] The DOX-GLU was conjugated with a targeting protein by drop-wiseaddition to a saline solution of transferrin (TRF). The TRF can beeither iron-free (apo-transferrin) or iron-saturated (holo-transferrin).The desired molar ratio of DOX to TRF was obtained by appropriatelyadjusting the volume of TRF. The resulting solution of TRF-GLU-DOX wasstirred for 20 hours at room temperature in the dark. Unlike thereaction of DOX with GLU, the reaction of DOX-GLU with TRF is notrestricted to one binding site, for the GLU component of DOX-GLU canreact with any one of several epsilon-amino lysine groups in the TRFmolecule.

[0052] The number of DOX molecules bound to TRF was determined bycalculation For example, if the starting ratio of DOX-GLU to TRF was7.2:1.0, the final solution of TRF-GLU-DOX would have contained 2.5molecules of DOX per molecule of TRF. However, if the staring ratio ofDOX-GLU to TRF was 4.0:1.0, the final solution of TRF-GLU-DOX would havecontained 1.4 molecules of DOX per molecule of TRF. Similarly, if thestaring ratio of DOX-GLU to TRF was 2.5:1.0, the final solution ofTRF-GLU-DOX would have contained 0.9 molecules of DOX per molecule ofTRF. In this way, large amounts of TRF-GLU-DOX with predetermined ratiosof DOX-to-TRF can be provided according to the need.

[0053] Further steps in the conjugation reaction were the addition ofethanolamine or another substance suitable for scavenging any excesslinker, followed by centrifugation and dialysis. Although reactions withDOX and TRF theoretically consume all of the GLU, ethanolamine was addedto the final reaction mixture to bind any available GLU. This reactionwas allowed to continue for 30 minutes in the dark The final solutionwas centrifuged at 2000 rpm for 10 minutes, dialyzed twice for 6 hoursin a 100-fold excess of saline and three times in the same excess ofHepes buffered saline, and the resulting TRF-GLU-DOX conjugates wereready for use.

[0054] Biochemical Characterization of the Conjugates:

[0055] By using HPLC and polyacrylamide gel electrophoresis as describedin (39), the homogeneity of TRF-GLU-DOX conjugates can be determined.Also, by using spectrophotometry as described in (89), the molecularratio of DOX-to-TRF can be determined. These techniques repeatedly haverevealed a consistent homogeneity of the TRF-GLU-DOX conjugates. Inaddition, chromatography is not required in the preparation of theseconjugates, because there are no. aggregates or fragments. This allowsfor the preparation of large volumes of homogeneous transferrin-drugconjugates, which increases yields and decreases costs.

[0056] The expenses caused by losses of TRF and DOX in other types oftransferrin-drug conjugates have been an impediment to their use. Forexample, yields of DOX and TRF are decreased by using procedures such asthiolation (44) that alter the drug and/or protein Yields also aredecreased by using solvent systems (86) and by chromatography used toprepare acid-stable and acid-labile linkages (41). The GLU bond betweenDOX and TRF is acid-stable (89), and yields of DOX and TRF in TRF-DOXconjugates prepared according to this invention are high. Indeed,compared to other procedures (38, 39, 40), the yield for TRF is nearlydoubled (90% vs 50%), and the yield for DOX is increased 5-fold.

[0057] None of the previously known approaches to the preparation oftransferrin-doxorubicin conjugates are capable of producing largeamounts of homogeneous conjugates with predetermined ratios of thenumber of drug molecules per molecule of transferrin. In addition, theother approaches employ chromatography to eliminate aggregates and toharvest fractions that are enriched in homogeneous conjugates. Theseprocedures decrease yields, increase costs, and lack the ability topredetermine molecular ratios.

[0058] Another procedure would be to mix one milliliter of transferrin(0.5 mM) with one milliliter of deferoxamine (8.5 mM) in 150 mM sodiumchloride for 4 minutes, and then add one milliliter of 21.5 mMglutaraldehyde in 150 mM sodium chloride and mix 4 minutes. Thepreceding reaction is a coupling procedure, which is stopped by theaddition of 0.8 milliliters of 37.2 mM ethanolamine in 150 mM sodiumchloride and 10 mM Hepes buffer (pH8) and vortexed for 4 minutes. Themixture (3.8 milers) then is transferred to dialysis tubing (molecularweight cutoff of 12,000-14,000), and dialyzed against 0.5 liters ofHepes-buffered saline in the dark at 5° C. for 3 hours. The dialysisshould be repeated at least once with fresh Hepes-buffered saline. Themixture then is centrifuged at 1600 g for 10 minutes at 4° C. and thesupernatant is chromatographed at a flow rate of 22 milliliters per houron a 2.6×34 cm column of Sepharose CL-4B, previously equilibrated inHepes-buffered saline and calibrated at 5° C. with blue dexran,transferrin and cytochrome C. Elution from the column is monitored at280 nm, and 3.8 milliliter fractions are collected. The concentration oftransferrin and deferoxamine in each fraction is calculated bysuccessive approximation from standard curves from transferrin anddeferoxamine, determined by using 280 nm for transferrin and 356 nm fordeferoxamine. With minor modifications, this coupling procedure can beused to prepare targeting protein conjugates of other iron chelatingdrugs, such as protein conjugates of hydrophobic reversed siderophores.

[0059] Characterizing the Conjugates

[0060] After the pure drug-protein conjugates are isolated, they arecharacterized by polyacrylamide gel electrophoresis to determine theirmolecular weight, and the number of drug molecules per protein moleculeis determined. The exact number of drug molecules per transferrinmolecule can be determined, using any suitable technique including butnot limited to spectrophotometric techniques. A functional drug:proteinratio is between about 0.1:1.0 to 3.0:1.0 (Berczi et al., Arch BiochemBiophys 1993; 300:356). The conjugates are checked to determine if theybind to receptors on the surface of tumor cells, and to determine if theconjugates kill cancer cells but not normal cells. Only conjugates thatbind to cancer cells and not to normal cells are selected for toxicitytests using drug-sensitive and drug-resistant cancer cells. The bindingstudies can be done by using flow cytometry or any other suitablemethod, and the killing studies can be done by using microculturetechniques to determine the concentration of free drug required to kill50% of a culture of cancer cells compared to the concentration of drugin the drug-protein conjugates required to kill the same number ofcancer cells. When testing the heat sensitizer conjugates, the toxicitytest is done by using the MIT tetrazolium colorimetric assay (Visiticaet al., Cancer Res 1994; 51:2515). These toxicity tests determine themost potent transferrin sensitizer ratio and the optimum concentrationof conjugate for maximum heat sensitization of drug sensitive and drugresistant cells. Approximately 10-fold more free drug compared to drugin the drug-protein conjugate is required to kill the same number ofcells.

[0061] While the above description refers to transferrin as being thedelivery protein, it is known that other proteins exist in the bodywhich are capable of binding to receptor sites on cells. If such areceptor site is activated in cancer cells and is inactive in normalcells, then any protein-or other molecule (ie., ligand) that binds tosuch a receptor site can be used to deliver the drugs used in thepresent invention. An example of such a binding protein istranscobalamin, which delivers vitamin B12 to transcobalamin receptorson cells, including cancer cells (Seetheram, Ann Rev Nutr 1999; 19:173).Low density lipoprotein is another ligand that has been conjugated tothe photosensitizer chlorin and targeted to low density lipoproteinreceptors on retinoblastoma cells (Schmidt-Erfurth et al., Brit J Surg1997; 75:54).

[0062] After the drug-protein conjugate has been prepared, purified,characterized and validated for cellular binding and killing properties,and, when the binding and killing experiments show that the conjugatebinds to and kills cancer but not normal cells, the conjugate is thenaliquoted and sterilized. The sterilization process can be done by anysuitable method including but not limited to exposure to irradiation,such as by using a cesium irradiator, or by using Millipore filtrationtechniques.

[0063] According to a further aspect of the present invention, there isprovided a reagent kit for the treatment of tumors, comprisingiron-bearing transferrin and a conjugate of transferrin with either aphotosensitizer, a heat sensitizer agent or an apoptosis inducingcompound. The patient's normal cells which have transferrin receptorsmay be protected against the effects of the conjugate by saturatingthese receptors with the iron-bearing transferrin before administrationof the photosensitizer, heat sensitizer or apoptosis inducingconjugates.

[0064] The present invention also provides a process for determining thesusceptibility of tumor cells to anti-tumor agents, comprisingadministering separately to portions of said tumor cells conjugates oftransferrin with a number of different photosensitizer, heat sensitizerand/or apoptosis inducing agents. A reagent kit comprising a number ofsuch different conjugates may be provided for this purpose. It has beenfound that tumor cells take up the conjugates of the present inventionextremely rapidly. This means that within a matter of hours of removalfrom the patient, tumor cells may be tested against a range ofconjugates of transferrin with different anti-tumor agents. Such aprocess would enable the chemotherapy which is most effective for agiven patient to be determined as soon as possible after isolation ofthe tumor cells.

[0065] The conjugates according to the present invention areadministered to an animal in an effective amount. In treating cancer, aneffective amount includes an amount effective to: reduce the size of atumor; slow the growth of a tumor; prevent or inhibit metastases; orincrease the life expectancy of the affected animal. The presentinvention provides for a method of treating cancers wherein the cancercan be but is not limited to a leukemia, breast cancer, ovarian cancer,pancreatic cancer, lung cancer, bladder cancer, gastrointestinal cancer,nasopharyngeal cancer, cervical cancer, sarcoma, myeloma,lymphoma/melanoma, glioma, or astrocytoma. The dosage for the conjugatescan be determined taking into account the age, weight and condition ofthe patient and the pharmacokinetics of the anti-tumor agent. The amountof the conjugate required for effective treatment will be less than theamount required using the anti-tumor agent alone.

[0066] The pharmaceutical compositions of the invention can beadministered by a number of routes, including but not limited to orally,topically, rectally, vaginally, by pulmonary route, for instance, by useof an aerosol, or parenterally, including but not limited tointramuscularly, subcutaneously, intraperitoneally, intra-arterially orintravenously. The compositions can be administered alone, or can becombined with a pharmaceutically-acceptable carrier or excipientaccording to standard pharmaceutical practice. For the oral mode ofadministration, the compositions can be used in the form of tablets,capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutionsand suspensions, and the like. For parenteral administration, sterilesolutions of the conjugate are usually prepared, and the pHs of thesolutions are suitably adjusted and buffered. For intravenous use, thetotal concentration of solutes should be controlled to render thepreparation isotonic. For ocular adminstration, ointments or droppableliquids may be delivered by ocular delivery systems known to the artsuch as applicators or eye droppers. For pulmonary administration,diluents and/or carriers will be selected to be appropriate to allow theformation of an aerosoL It is preferred that the conjugate of thepresent invention be administered parenterally, i.e. intravenously orintraperitoneally, by infusion or injection.

[0067] Preferred embodiments of the present invention are describedbelow. It will be apparent to those of ordinary skill in the art afterreading the following description that modifications and variations arepossible, all of which are intended to fall within the scope of theclaims.

EXAMPLE 1

[0068] Preparation of a Homogeneous Transferrin-Doxorubicin Conjugate

[0069] The synthesis of large amounts of homogeneoustransferrin-doxorubicin conjugates with predetermined molecular ratioswas done stoichiometrically by employing the only amino group ofdoxorubicin (DOX), which is at the 3′ amino position, to react with oneof the two reactive groups on glutaraldehyde (GLU). The first step wasto add GLU drop-wise to DMSO in an ice cold water bath Next was thedrop-wise addition of a saline solution of DOX into a saline solution ofGLU+DMSO to a final concentration of a 1:1 molar ratio of DOX-to-GLU.The resulting solution of DOX-GLU was stirred three hours at roomtemperature in the dark.

[0070] The molarities of DOX and GLU were the same in the above reactionin order to produce a final solution of DOX-GLU that contains neitherfree DOX nor free GLU. However, there is the possibility of free GLU insolution if one GLU reacts with two DOX to produce DOX-GLU-DOX, but thispossibility is minimized by the mass action kinetics generated bydrop-wise addition of monovalent DOX into the solution of bivalent GLU.The volumes of these reactants are not restricted, so large amounts ofhomogeneous DOX-GLU can be prepared.

[0071] Subsequently DOX-GLU was added to transferrin (TRF) by drop-wiseaddition into a saline solution of the transferrin (TRF). The TRF can beeither iron-free (apo-transferrin) or iron-saturated (holo-transfernin).The desired molar ratio of DOX to TRF was obtained by appropriatelyadjusting the volume of TRF. The resulting solution of TRF-GLU-DOX wasstirred for 20 hours at room temperature in the dark Unlike the reactionof DOX with GLU, the reaction of DOX-GLU with TRF is not restricted toone binding site, for the GLU component of DOX-GLU can react with anyone of several epsilon-amino lysine groups in the TRF molecule.

[0072] The number of DOX molecules bound to TRF was determined in thesecond step. For example, if the starting ratio of DOX-GLU to TRF was7.2:1.0, the final solution of TRF-GLU-DOX would have contained 2.5molecules of DOX per molecule of TRF. However, if the starting ratio ofDOX-GLU to TRF was 4.0:1.0, the final solution of TRF-GLU-DOX would havecontained 1.4 molecules of DOX per molecule of TRF. Similarly, if thestarting ratio of DOX-GLU to TRF was 2.5:1.0, the final solution ofTRF-GLU-DOX would have contained 0.9 molecules of DOX per molecule ofTRF. In this way, large amounts of TRF-GLU-DOX with predetermined ratiosof DOX-to-TRF can be provided according to the need.

[0073] Any excess GLU remaining in the reaction product can be scavengedby the addition of ethanolamine, followed by centrifigation anddialysis. Although reactions with DOX and TRF theoretically consume allof the GLU, ethanolamine was added to the final reaction mixture to bindany available GLU. This reaction was allowed to continue for 30 minutesin the dark. The final solution was centrifuged at 2000 rpm for 10minutes, dialyzed twice for 6 hours in a 100-fold excess of saline andthree times in the same excess of Hepes buffered saline, and theresulting TRF-GLU-DOX conjugates were ready for use.

[0074] Biochemical Characterization of the Conjugates.

[0075] By using HPLC and polyacrylamide gel electrophoresis, thehomogeneity of TRF-GLU-DOX conjugates can be determined Also, by usingspectrophotometry, the molecular ratio of DOX-to-TRF can be determined.These techniques repeatedly have revealed a consistent homogeneity ofthe TRF-GLU-DOX conjugates. In addition, chromatography is not requiredin the preparation of these conjugates, because there are no aggregatesor fragments. This allows for the preparation of large volumes ofhomogeneous transferrin-drug conjugates, which increases yields anddecreases costs.

[0076] The expenses caused by losses of TRF and DOX in other types oftransferrin-drug conjugates have been an impediment to their use. Forexample, yields of DOX and TRF are decreased by using procedures such asthiolation that alter the drug and/or protein. Yields also are decreasedby using solvent systems and by chromatography used to prepareacid-stable and acid-labile linkages. The GLU bond between DOX and TRFis acid-stable, and yields of DOX and TRF in TRF-DOX conjugates preparedaccording to this invention are high Indeed, compared to other knownprocedures, the yield for TRF is nearly doubled (90% vs 50%), and theyield for DOX is increased 5-fold.

[0077] None of the previously known approaches to the preparation oftransferrin-doxorubicin conjugates are capable of producing largeamounts of homogeneous conjugates with predetermined ratios of thenumber of drug molecules per molecule of transferring. In addition, theother approaches employ chromatography to eliminate aggregates and toharvest fractions that are enriched in homogeneous conjugates. Theseprocedures decrease yields, increase costs, and lack the ability topredetermine molecular ratios.

Example 2

[0078] Preparation of a Transferrin-Apoptosis Inducing Compound(Deferoxamine) Conjugate

[0079] Glutaraldehyde (GLU) is added drop-wise to DMSO in an ice coldwater bath followed by the drop-wise addition of a saline solution ofdeferoxamine (DEF) into a saline solution of GLU+DMSO to a finalconcentration of a 2:1 molar ratio of DEF-to-GLU. The resulting solutionof DEF-GLU is stirred three hours at room temperature in the dark.

[0080] The DEF-GLU is added drop-wise to a saline solution oftransferrin (TRF). The TRF can be either iron-free (apo-transferrin) oriron-saturated (holo-transferrin). The desired molar ratio of DEF to TRFis obtained by appropriately adjusting the volume of TRF. The resultingsolution of TRF-GLU-DEF was stirred for 20 hours at room temperature inthe dark. Unlike the reaction of DEF with GLU, the reaction of DEF-GLUwith TRE is not restricted to one binding site, for the GLU component ofDEF-GLU can react with any one of several epsilon-amino lysine groups inthe TRF molecule.

[0081] The number of DEF molecules bound to TRF is determined during theaddition of DEF-GLU to TRF. For example, if the starting ratio ofDEF-GLU to TRF was 7.2:1.0, the final solution of TRF-GLU-DEF would havecontained 2.5 molecules of DEF per molecule of TRF. However, if thestarting ratio of DEF-GLU to TRF is 4.0:1.0, the final solution ofTRF-GLU-DEF would have contain 1.4 molecules of DEF per molecule of TRF.Similarly, if the starting ratio of DEF-GLU to TRF is 2.5:1.0, the finalsolution of TRF-GLU-DEF would contain 0.9 molecules of DEF per moleculeof TRF. In this way, large amounts of TRF-GLU-DEF with predeterminedratios of DEF-to-TRF can be provided according to need.

[0082] Ethanolamine is normally added to the reaction product followedby centrifugation and dialysis. Although reactions with DEF and TRFtheoretically consume all of the GLU, ethanolamine is added to the finalreaction mixture to bind any available GLU. This reaction is allowed tocontinue for 30 minutes in the dark. The final solution is centrifugedat 2000 rpm for 10 minutes, dialyzed twice for 6 hours in a 100-foldexcess of saline and three times in the same excess of Hepes bufferedsaline, and the resulting TRF-GLU-DEF conjugates are ready for use.

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1. A substantially homogeneous conjugate comprising a targeting agentand a bioaffecting material, wherein said bioaffecting material isselected from the group consisting of a photosensitizer, a heatsensitizer and an apoptosis inducing compound and said targeting agentis selected from the group consisting of transfernin, somatostatin,epidermal growth factor, folic acid and transcobalamin, and wherein saidhomogeneous conjugate is substantially free of dimers, trimers andaggregates.
 2. The conjugate according to claim 1, wherein saidtargeting agent is transferrin.
 3. The conjugate according to claim 1,wherein said bioaffecting material is a photosensitizer.
 4. Theconjugate according to claim 3, wherein said photosensitizer is selectedfrom the group consisting of porfimer sodium, aminolevulinic acid,metatetrahydroxyphenylchlorin and a haematophorphyrin derivative.
 5. Theconjugate according to claim 1, wherein said bioaffecting material is aheat sensitizer.
 6. The conjugate according to claim 5, wherein saidheat sensitizer is selected from the group consisting ofhaematophorphyrin, verapamil bleomycin, carboplatin, doxorubicin, andcisplatin.
 7. The conjugate according to claim 1, wherein saidbioaffecting material is an apoptosis inducing compound.
 8. Theconjugate according to claim 7, wherein said apoptosis inducing compoundis deferoxamine.
 9. A method for making a conjugate having apredetermined drug: protein ratio, comprising a) adding a solution of adrug dropwise to a molar excess of a linker molecule solution to linkeach drug molecule to one linker molecule in a drug/linker combination;wherein when said drug molecule is deferoxamine two drug molecules areattached to one linker molecule and b) adding the drug/linkercombination to a protein to produce a conjugate having a predetermineddrug protein ratio, wherein said drug is selected from the groupconsisting of a heat sensitizer, a photosensitizer and an apoptosisinducing compound.
 10. The method according to claim 9, furthercomprising scavenging any excess linker.
 11. The method according toclaim 9, wherein said linker is selected from the group consisting ofglutaraldehyde, benzoyl hydrazone, maleinitide and N-hydroxysuccinimide.12. The method according to claim 9, wherein said drug is selected fromthe group consisting of porfimer sodium, aminolevulinic acid,metatetrahydroxyphenylchlorin, haematophorphyrin derivative,hematophosphyrine, verapanil, doxornbicin, cisplatin, and deferoxamine.13. A reagent kit for the treatment of diseased cells, comprisingiron-bearing transferrin, and a conjugate containing a protein targetingagent and a bioaffecting material, wherein said bioaffecting material isselected from the group consisting of heat sensitizers, photosensitizersand apoptosis inducing compounds.
 14. A reagent kit for determining thesusceptibility of tumor cells to anti-tumor agents, comprising two ormore conjugates each containing a protein targeting agent and ananti-tumor drug, wherein said anti-tumor drug is selected from the groupconsisting of heat sensitizers, photosensitizers and apoptosis inducingcompounds, and wherein said conjugates have different antitumor drugs.15. A method for selectively treating a tumor susceptible tohyperthermia therapy, comprising administering to a patient with saidtumor an anti-tumor effective amount of the conjugate according to claim1, wherein said anti-cancer agent is a heat sensitizer, and producing acontrolled temperature increase in said tumor.
 16. The method accordingto claim 15, wherein said heat sensitizer is selected from the groupconsisting of haematophorphyrin, verapamil, bleomycin, carboplatin,doxorubicin, and cisplatin.
 17. The method according to claim 15,wherein said controlled temperature increase is produced by exposing thetumor to YAG lasers, radio frequencies or utrasound.
 18. A method forselectively treating a diseased cell susceptible to photodynamictherapy, comprising administering to a patient with a tumor ananti-tumor effective amount of the conjugate according to claim 1,wherein said anti-cancer agent is a photosensitizer, and exposing saidtumor to a suitable wavelength of light to activate saidphotosensitizer.
 19. The method according to claim 18, wherein saidphotosensitizer is selected from the group consisting of porfimersodiimn, aminolevulinic acid, metatetrahydroxyphenylchlorin and ahaematophorphyrin derivative.
 20. A method for selectively treating atumor susceptible to iron restriction therapy, comprising administeringto a patient with a tumor an anti-tumor effective amount of theconjugate according to claim 1, wherein said anti-cancer agent is anapoptosis inducing compound which restricts iron.
 21. The methodaccording to claim 20, further comprising administering free iron ortransferrin bound iron to said patient.
 22. The method according toclaim 20, wherein said apoptosis inducing compound which restricts ironis deferoxmine.
 23. A method for selectively treating target cells,comprising contacting target cells with the conjugate according to claim1, wherein said anti-cancer agent is an apoptosis inducing compound. 24.The method according to claim 23, further comprising administering freeiron or transferrin bound iron to said target cells.
 25. A method forselectively treating drug resistant target cells comprising contactingtarget cells with the conjugate according to claim 1, wherein saidanti-cancer agent is selected from the group consisting of aphotosensitizer and a heat sensitizer, and exposing said target cells toa controlled temperature increase or a suitable wavelength of light.